Investigating the Effect of Low-Level Water Addition for Preparative Chiral SFC

Preparative chiral supercritical fluid chromatography (SFC) is widely used in pharmaceutical development to separate and purify enantiomers, but even small improvements in chromatographic performance can have a meaningful impact on productivity, solvent consumption, and process efficiency. Previous analytical-scale studies have suggested that adding small amounts of water to the mobile phase can improve separation performance, yet the potential benefits and limitations of this approach under preparative chiral SFC conditions have received considerably less attention.

In a recent study, researchers systematically investigated the effect of low-level water addition across ten preparative chiral stationary phases representing a broad range of chemistries. Water concentrations of 0%, 2.5%, 5%, and 7.5% (v/v) were evaluated to determine how this simple mobile-phase modification affected retention, peak shape, and collection windows. Across most of the columns examined, adding water reduced retention times and narrowed collection windows, with much of the benefit occurring at relatively low concentrations.

The study also revealed important differences between stationary phases, including an unexpected non-monotonic response for S,S-Whelk-O1. LCGC International spoke with Xiaochun (Alex) Wang from Pfizer Research and Development about the mechanisms behind these effects, the role of stationary-phase chemistry, and the practical implications for improving throughput, loading capacity, and solvent efficiency in preparative chiral SFC.

Why did your team investigate the effect of low-level water addition in preparative chiral supercritical fluid chromatography (SFC)?

Xiaochun (Alex) Wang: One of the primary motivations for this work was the observation that several analytical-scale SFC studies had reported improved chromatographic performance with the addition of small amounts of water,^1–5 yet very little information was available regarding its impact under preparative chiral SFC conditions. Preparative SFC is widely used in the pharmaceutical industry for chiral purification, where even modest improvements in retention, peak shape, or loading capacity can have a substantial impact on throughput, cost, and overall process efficiency.

We wanted to determine whether the benefits observed at the analytical scale would translate to preparative applications. To address this question, we systematically evaluated low-level water addition across a diverse set of preparative chiral stationary phases. Our findings demonstrated that even small amounts of water can significantly reduce retention times and narrow collection windows, highlighting a straightforward and practical strategy for improving preparative separations.^1,6

Why did the researchers evaluate water concentrations of 0%, 2.5%, 5%, and 7.5% (v/v), and how do these levels reflect practical operating conditions in preparative chiral SFC?

XW: These concentrations were chosen because they are readily achievable in preparative chiral SFC and align with conditions previously explored in analytical SFC studies. Our focus was on investigating the effects of low-level water addition, where water remains a minor component of the alcohol modifier. From an operational standpoint, these compositions are practical because water can be incorporated into the alcohol modifier to form a CO₂/alcohol/H₂O ternary mobile phase before phase separation.

An important observation was that much of the chromatographic benefit occurred with the initial addition of water. Adding only 2.5% water produced an immediate reduction in retention on most columns, while increasing the concentration to 5% and 7.5% provided further, though often smaller, decreases in retention. These results suggest that relatively small amounts of water are sufficient to achieve substantial chromatographic improvements, making the 2.5% to 5% range especially attractive for preparative applications.

The study examined ten different preparative chiral columns with varying stationary-phase chemistries. What was the purpose of including such a diverse range of stationary phases, and how did this strengthen the study's conclusions?

XW: We intentionally selected a broad range of stationary-phase chemistries to ensure that our conclusions would be broadly relevant rather than specific to a single column type. The study included seven polysaccharide-derived phases, one Pirkle-type phase, and two macrocyclic glycopeptide phases, representing the major classes commonly used for chiral separations.

By evaluating multiple stationary phases, we were able to determine whether the effect of water was a general phenomenon or dependent on a particular chiral selector. The fact that nine of the ten columns exhibited similar overall trends strongly supports the conclusion that low-level water addition can be beneficial across a wide range of preparative chiral SFC applications.

The authors found that adding low levels of water generally reduced retention times and narrowed collection windows for most columns. What mechanisms could explain these improvements in chromatographic performance?

XW: Several mechanisms may contribute to the improvements we observed.

First, water likely adsorbs to polar sites on the stationary phase and competes with analytes for those interactions. This competition reduces strong analyte–stationary-phase interactions, particularly hydrogen-bonding and other polar interactions, allowing compounds to elute more rapidly.

Second, water may form a thin water-rich layer on the stationary phase, similar to what is observed in hydrophilic interaction liquid chromatography (HILIC). Such a layer can modify retention behavior, reduce analyte adsorption, and improve mass transfer, leading to narrower peaks and improved efficiency.

Third, water can improve the solubility of certain analytes in the mobile phase. Enhanced solubility may reduce band broadening and potentially increase loading capacity under preparative conditions.

Collectively, these effects result in faster analyte desorption, reduced retention, improved peak shape, and narrower collection windows. For preparative chromatography, that translates directly into higher throughput, smaller fraction volumes, and more efficient purifications.

Were there any results that surprised you?

XW: Yes. The most surprising result involved the S,S-Whelk-O1 stationary phase.

While nine of the ten columns showed the expected trend of decreasing retention with increasing water content, S,S-Whelk-O1 displayed a non-monotonic response. Retention initially decreased at lower water concentrations but then increased as additional water was added. We observed this behavior across multiple analytes, suggesting that it was an intrinsic property of the stationary phase rather than a compound-specific effect.

Another unexpected finding was the relatively weak relationship between stationary-phase hydrophilicity and the magnitude of the water effect. We anticipated that the most hydrophilic phases would exhibit the largest responses; however, the data revealed a more complex interplay of chromatographic interactions than hydrophilicity alone could explain.

The researchers used hydrophilic interaction liquid chromatography (HILIC) experiments to help interpret their SFC results. Why was HILIC chosen as a complementary approach, and how did it help explain the observed effects of water addition?

XW: HILIC was selected because several of the proposed mechanisms for water in SFC involve hydrophilic interactions and the formation of a water-rich layer on the stationary phase, concepts that are well established in HILIC.

By conducting HILIC experiments and examining retention behaviour as a function of water content, we gained additional insight into how each stationary phase interacted with water. These studies allowed us to assess relative stationary-phase hydrophilicity and provided mechanistic context for the retention changes observed in SFC.

Importantly, the HILIC results also helped explain the unusual behaviour of S,S-Whelk-O1. The data suggested that this stationary phase relies on a different balance of polar, hydrophobic, and π–π interactions compared with the polysaccharide and glycopeptide phases, leading to a different response as water content increased.

Based on the findings of this study, what are the potential practical implications for pharmaceutical purification using preparative chiral SFC, and what further research would you propose to expand on these results?

XW: From a practical perspective, our results suggest that adding a small amount of water, particularly in the range of 2% to 5%, can be an effective and straightforward optimization strategy for preparative chiral SFC. Across most stationary phases, water addition reduced retention times and narrowed collection windows, resulting in potential gains in throughput, peak efficiency, solvent savings, and loading capacity.

One of the most compelling examples from the study demonstrated that the addition of 5% water enabled a tenfold increase in loading while reducing both the number of injections required and the total solvent consumption. For preparative laboratories, these improvements can translate into meaningful gains in productivity and cost efficiency.

Looking ahead, several areas warrant further investigation. More extensive studies of loading capacity across diverse compound classes would be valuable. Long-term evaluations of column stability under routine water-containing SFC conditions are also needed. In addition, further mechanistic studies could provide deeper insight into how water interacts with different classes of chiral selectors. Finally, expanding this approach to more challenging pharmaceutical separations would help establish broader guidelines for implementation in preparative workflows.

Anything you would like to add?

XW: As separation scientists, we're always looking for practical ways to improve chromatography. What excited me most about this work was seeing how the simple addition of a small amount of water could dramatically improve performance across a wide range of preparative chiral columns. I hope these findings provide useful insights for scientists working in both industry and academia, and I look forward to seeing how the community builds on this work in the future.

References

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3. Bennett, R.; Biba, M.; Liu, J.; et al. Enhanced Fluidity Liquid Chromatography: A Guide to Scaling Up from Analytical to Preparative Separations. J Chromatogr A 2019, 1595, 190−198. DOI: 10.1016/j.chroma.2019.02.017
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6. Wang, X.; Bradow, J.; Smith, J.; Li, C. Characterization of Low-level Water Addition for Preparative Chiral SFC. J Chromatogr A 2026, 1783, 467200. DOI: 10.1016/j.chroma.2026.467200